Detection of Mycoplasma in Cell Cultures and Cell Culture derived Biologicals

• US 20150111207A1
• Filed: 05/31/2013
• Published: 04/23/2015
• Est. Priority Date: 06/01/2012
• Status: Active Grant

• Current Assignee: Akron Biotechnology, LLC
• Original Assignee: Akron Biotech
• Inventors: Zylberberg, Claudia, Rodriguez, Juan Manuel, Hernando Insua, Andres Clemente, Lopez, Ricardo Ariel
• Granted Patent: US 10,472,683 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Sequences having specificity for Mycoplasma and related Mollicutes genus strains and uses thereof. Methods of use include detection of samples contaminated with Mycoplasma. Kits are provided and comprise one or more oligonucleotides for the detection of Mycoplasma and related Mollicutes genus strains.

Claims

1. An isolated or synthetic nucleic acid, wherein the isolated or synthetic nucleic acid hybridizes to at least 5 consecutive nucleic acids in a 5′

to 3′

or 3′

to 5′

direction of a 16S-23S ribosomal intergenic region nucleic acid of Mycoplasma and/or related Mollicutes genus strains.

2. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid comprises at least five nucleic acids.

3. The isolated nucleic acid of claim 1, optionally comprising one or more mutations, variants, substitutions, analogs, deletions or combinations thereof.

4. The isolated nucleic acid of claim 3, wherein the one or more mutations, variants, substitutions, analogs or combinations thereof, comprise:

adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine, N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine, non-naturally occurring nucleobases, locked nucleic acids (LNA), or peptide nucleic acids (PNA).

5. The isolated nucleic acid of claim 1, wherein the Mycoplasma and/or related Mollicutes genus strains, comprising:

Acholeplasma laidlawii ATCC23206;

Spiroplasma citri ATCC27556;

M. arginini ATCC23838;

M. arthritidis ATCC19611;

M. bovis ATCC25025;

M. capricolum capricolum ATCC27343;

M. fermentans ATCC49892;

M. gallisepticum ATCC19610;

M. genitalium ATCC33530;

M. hominis ATCC27545;

M. hyopneumoniae ATCC25934;

M. orale ATCC23714;

M. pirum ATCC25960;

M. pneumoniae ATCC15531;

M. pulmonis GenBank X58554;

M. salivarium ATCC23064;

M. synoviae ATCC25204;

M. mirounga GenBank GU905026;

M. adleri strain G145;

M. agalactiae strainGO-5;

M. agassizii strain PS6;

M. alkalescens ATCC29103;

M. alligatoris ATCC700619;

M. alvi strain Ilsley;

M. amphoriforme strain A39;

M. anseris strain 1219;

M. auris strain UTA;

M. bovigenitalium strain PG-11;

M. bovirhinis ATCC27748;

M. bovoculi ATCC29104;

M. buccale ATCC23636;

M. buteonis strain Bb/T2g;

M. californicum strain ST6;

M. canadense strain 466;

M. canimucosale type strain 1642;

M. canis ATCC19525;

M. caviae strain ATCC27108;

M. cavipharyngis ATCC43016;

M. citelli strain RG2C;

M. cloacale strain 383;

M. collis ATCC35278;

M. columbinasale ATCC33549;

M. columbinum strain FG295;

M. columborale ATCC29258;

M. conjunctivae Goat655;

M. corogypsi strain BV1-5;

M. cottewii ATCC43093;

M. cricetuli ATCC35279;

M. crocodyli ATCC51981;

M. cynos strain H381;

M. dispar ATCC27140;

M. edwardii strain 04-3440;

M. elephantis ATCC51980;

M. equigenitalium ATCC29869;

M. equirhinis ATCC29420;

M. falconis ATCC51372;

M. fastidiosum ATCC33229;

M. faucium ATCC25293;

M. feliminutum ATCC25749;

M. felis GenBank AF443608;

M. flocculare strain Ms42;

M. gallinarum strain B2Dinter;

M. gallisepticum GenBank FJ468427;

M. gallopavonis ATCC33551;

M. glycophilum ATCC35277;

M. gypis ATCC51370;

M. hyopharyngis strain H36B-F;

M. hyorhinis ATCC29052;

M. iguanae strain 2327;

M. indiense ATCC51125;

M. iners ATCC15969;

M. iowae ATCC33552;

M. lagogenitalium ATCC700289;

M. leachii strain PG50;

M. leonicaptivi ATCC49890;

M. leopharyngis ATCC49889;

M. lipofaciens ATCC35015;

M. lipophilum ATCC27104;

M. maculosum strain PG15;

M. meleagridis ATCC27764;

M. microti strain IL371;

M. moatsii strain MK405;

M. mobile ATCC43663;

M. molare strain H542;

M. muris ATCC33757;

M. mustelae ATCC35214;

M. neurolyticum ATCC19988;

M. opalescens strain MHS408;

M. ovipneumoniae ATCC29419;

M. oxoniensis ATCC49694;

M. phocicerebrale strain CSL5195S2;

M. phocirhinis strain CSL7475-4;

M. primatum strain HRC292;

M. pullorum ATCC33553;

M. putrefaciens ATCC33756;

M. simbae ATCC49888;

M. spermatophilum strain AH159;

M. sphenisci strain UCMJ;

M. spumans ATCC19526;

M. sturni ATCC51945;

M. sualvi ATCC33004;

M. subdolum ATCC29870;

M. testudineum ATCC700618;

M. testudinis ATCC43263;

M. verecundum ATCC27862;

M. vulturii strain Gb-V33;

M. yeatsii ATCC43094;

M. zalophi strain CSL5195;

or M. zalophidermidis strain CSL4779.

6. The isolated nucleic acid of claim 1, wherein the nucleic acid hybridizes to the 16S-23S ribosomal intergenic region of Mycoplasma and related Mollicutes genus strains, under low-, medium- or high stringency conditions.

7. A nucleic acid sequence wherein the nucleic acid sequence is complementary to at least five consecutive nucleic acids in a 5′

to 3′

or 3′

to 5′

direction of a 16S-23S ribosomal intergenic region nucleic acid of Mycoplasma and/or related Mollicutes genus strains.

8. The nucleic acid sequence of claim 7, wherein the Mycoplasma and/or related Mollicutes genus strains, comprising:

Acholeplasma laidlawii ATCC23206;

Spiroplasma citri ATCC27556;

M. arginini ATCC23838;

M. arthritidis ATCC19611;

M. bovis ATCC25025;

M. capricolum capricolum ATCC27343;

M. fermentans ATCC49892;

M. gallisepticum ATCC19610;

M. genitalium ATCC33530;

M. hominis ATCC27545;

M. hyopneumoniae ATCC25934;

M. orale ATCC23714;

M. pirum ATCC25960;

M. pneumoniae ATCC15531;

M. pulmonis GenBank X58554;

M. salivarium ATCC23064;

M. synoviae ATCC25204;

M. mirounga GenBank GU905026;

M. adleri strain G145;

M. agalactiae strainGO-5;

M. agassizii strain PS6;

M. alkalescens ATCC29103;

M. alligatoris ATCC700619;

M. alvi strain Ilsley;

M. amphoriforme strain A39;

M. anseris strain 1219;

M. auris strain UTA;

M. bovigenitalium strain PG-11;

M. bovirhinis ATCC27748;

M. bovoculi ATCC29104;

M. buccale ATCC23636;

M. buteonis strain Bb/T2g;

M. californicum strain ST6;

M. canadense strain 466;

M. canimucosale type strain 1642;

M. canis ATCC19525;

M. caviae strain ATCC27108;

M. cavipharyngis ATCC43016;

M. citelli strain RG2C;

M. cloacale strain 383;

M. collis ATCC35278;

M. columbinasale ATCC33549;

M. columbinum strain FG295;

M. columborale ATCC29258;

M. conjunctivae Goat655;

M. corogypsi strain BV1-5;

M. cottewii ATCC43093;

M. cricetuli ATCC35279;

M. crocodyli ATCC51981;

M. cynos strain H381;

M. dispar ATCC27140;

M. edwardii strain 04-3440;

M. elephantis ATCC51980;

M. equigenitalium ATCC29869;

M. equirhinis ATCC29420;

M. falconis ATCC51372;

M. fastidiosum ATCC33229;

M. faucium ATCC25293;

M. feliminutum ATCC25749;

M. felis GenBank AF443608;

M. flocculare strain Ms42;

M. gallinarum strain B2Dinter;

M. gallisepticum GenBank FJ468427;

M. gallopavonis ATCC33551;

M. glycophilum ATCC35277;

M. gypis ATCC51370;

M. hyopharyngis strain H36B-F;

M. hyorhinis ATCC29052;

M. iguanae strain 2327;

M. indiense ATCC51125;

M. iners ATCC15969;

M. iowae ATCC33552;

M. lagogenitalium ATCC700289;

M. leachii strain PG50;

M. leonicaptivi ATCC49890;

M. leopharyngis ATCC49889;

M. lipofaciens ATCC35015;

M. lipophilum ATCC27104;

M. maculosum strain PG15;

M. meleagridis ATCC27764;

M. microti strain IL371;

M. moatsii strain MK405;

M. mobile ATCC43663;

M. molare strain H542;

M. muris ATCC33757;

M. mustelae ATCC35214;

M. neurolyticum ATCC19988;

M. opalescens strain MHS408;

M. ovipneumoniae ATCC29419;

M. oxoniensis ATCC49694;

M. phocicerebrale strain CSL5195S2;

M. phocirhinis strain CSL7475-4;

M. primatum strain HRC292;

M. pullorum ATCC33553;

M. putrefaciens ATCC33756;

M. simbae ATCC49888;

M. spermatophilum strain AH159;

M. sphenisci strain UCMJ;

M. spumans ATCC19526;

M. sturni ATCC51945;

M. sualvi ATCC33004;

M. subdolum ATCC29870;

M. testudineum ATCC700618;

M. testudinis ATCC43263;

M. verecundum ATCC27862;

M. vulturii strain Gb-V33;

M. yeatsii ATCC43094;

M. zalophi strain CSL5195;

or M. zalophidermidis strain CSL4779.

9. A nucleic acid sequence, wherein the nucleic acid sequence is complementary to at least five consecutive nucleic acids in a 5′

to 3′

or 3′

to 5′

direction, of any one or more sequences set forth as SEQ ID NOS;

1 to 15.

10. The nucleic acid sequence of claim 9, wherein the nucleic acid comprises at least five nucleic acids.

11. The nucleic acid of claim 10, optionally comprising one or more mutations, variants, substitutions, analogs, deletions or combinations thereof.

12. The nucleic acid of claim 11, wherein the nucleic acid comprises one, or combinations thereof, of:

adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine, N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine, non-naturally occurring nucleobases, locked nucleic acids (LNA), or peptide nucleic acids (PNA).

13. An isolated nucleic acid, wherein a nucleic acid sequence comprises a sequence identity of at least about 50% to about 99.99% to at least five consecutive nucleic acids, or complementary sequences thereof, in either a sense or anti-sense orientation, of a 16S-23S ribosomal intergenic of Mycoplasma and related Mollicutes genus strains.

14. A nucleic acid sequence comprising a mutated Mycoplasma sequence wherein the sequence is a discriminatory positive control (DPC) and comprises at least one sequence set forth as SEQ ID NOS:

1 to 9, mutants, variants and fragments thereof.

15. An oligomer comprising a nucleic acid sequence complementary to at least five consecutive nucleic acids in a 5′

to 3′

or 3′

to 5′

direction, of any one or more sequences set forth as SEQ ID NOS;

1 to 9.

16. The oligomer of claim 15, wherein the nucleic acid sequence comprises at least five nucleic acids.

17. The oligomer of claim 15, optionally comprising one or more mutations, variants, substitutions, analogs, deletions or combinations thereof.

18. The oligomer of claim 17, wherein the nucleic acid comprises at least one of:

adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine, N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine, non-naturally occurring nucleobases, locked nucleic acids (LNA), peptide nucleic acids (PNA), or combinations thereof.

19. A nucleic acid sequence set forth as SEQ ID NOS:

1 to 15 or fragments thereof, comprising one or more nucleic acid base mutant, variants, analogs, deletions, substitutions or combinations thereof.

20. The nucleic acid sequence of claim 19, wherein the nucleic acid base mutant, variants, analogs, or substitutions comprise:

adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine, N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine, non-naturally occurring nucleobases, locked nucleic acids (LNA), peptide nucleic acids (PNA), or combinations thereof.

21. A method of detecting whether a sample is contaminated with Mycoplasma and/or related Mollicutes genus strains comprising:

incubating a sample with one or more primers or oligomers comprising nucleic acid sequences set forth as SEQ ID NOS;

1 to 15;

assaying the samples;

thereby,

detecting whether the sample is contaminated with Mycoplasma and/or related Mollicutes genus strains.

22. The method of claim 21, wherein incubating the samples further comprises incubating the samples with at least one discriminatory positive control set forth as SEQ ID NOS:

1 to 9.

23. The method of claim 21, wherein methods of assaying the samples comprise, polymerase chain reaction (PCR), immunoassays, blots, gels, microarrays, high-throughput screening assays, or combinations thereof.

24. The method of claim 23, wherein the PCR is a real-time polymerase chain reaction (RT-PCR).

25. A Mycoplasma detection kit comprising:

at least one oligomer comprising a nucleic acid sequence set forth as SEQ ID NOS;

1 to 15, and/or complementary sequences thereof.

26. The Mycoplasma detection kit of claim 25, further comprising buffers.

27. The Mycoplasma detection kit of claim 25, wherein the kit optionally comprises a negative control, primer solutions, nucleobases, dyes or combinations thereof.

28. An oligomer or primer, wherein oligomer or primer comprises a sequence identity of at least about 50% to any one nucleic acid sequence set forth as SEQ ID NOS:

1 to 15 in either a sense or anti-sense orientation or the 5′

to 3′

or 3′

to 5′

direction.

29. The oligomer or primer of claim 28, wherein the oligomer or primer comprises a sequence set forth in SEQ ID NOS:

1 to 15.

30. An oligomer comprising a sequence set forth as SEQ ID NOS:

1 to 9.

31. An oligomer comprising a sequence having at least a 50% sequence identity with either strand in the sense or anti-sense orientation of the sequences forth as SEQ ID NOS:

1 to 9.

32. An oligomer comprising a sequence set forth as SEQ ID NOS:

10 to 15.

33. An oligomer comprising a sequence having at least a 50% sequence identity with either strand in the sense or anti-sense orientation of the sequences forth as SEQ ID NOS:

10 to 15.

34. An oligomer comprising a sequence having at least a 50% sequence identity of the sequences forth as SEQ ID NOS:

1 to 15, mutants, variants, or complementary sequences thereof.

35. The oligomer of claim 34, further comprising one or more detectable labels.

36. A method of detecting whether a sample is contaminated with Mycoplasma and/or related Mollicutes genus strains comprising:

incubating a sample with two or more nucleic acid sequences set forth as SEQ ID NOS;

1 to 15;

assaying the samples;

thereby,

detecting whether the sample is contaminated with Mycoplasma and/or related Mollicutes genus strains

37. The method of claim 36, wherein one of the nucleic acid sequences is a discriminatory positive control set forth as SEQ ID NOS:

1 to 9.

38. The method of claim 36, wherein methods of assaying the samples comprise, polymerase chain reaction (PCR), immunoassays, blots, gels, microarrays, high-throughput screening assays, or combinations thereof.

39. The method of claim 38, wherein the PCR is a real-time polymerase chain reaction (RT-PCR).